Oxidative damage and myofiber degeneration in the gastrocnemius of patients with peripheral arterial disease.

Peripheral arterial disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in arteries supplying the legs, affects an estimated 27 million people in Europe and North America. Increased production of reactive oxygen species by dysfunctional mitochondria in leg muscles of PAD patients is viewed as a key mechanism of initiation and progression of the disease. Previous studies demonstrated increased oxidative damage in homogenates of biopsy specimens from PAD gastrocnemius compared to controls, but did not address myofiber-specific damage. In this study, we investigated oxidative damage to myofibers as a possible cause of the myopathy of PAD. To achieve this, we developed and validated fluorescence microscopy procedures for quantitative analysis of carbonyl groups and 4-hydroxy-2-nonenal (HNE) adducts in myofibers of biopsy specimens from human gastrocnemius. PAD and control specimens were evaluated for differences in 1) myofiber content of these two forms of oxidative damage and 2) myofiber cross-sectional area. Furthermore, oxidative damage to PAD myofibers was tested for associations with clinical stage of disease, degree of ischemia in the affected leg, and myofiber cross-sectional area. Carbonyl groups and HNE adducts were increased 30% (p < 0.0001) and 40% (p < 0.0001), respectively, in the myofibers of PAD (N = 34) compared to control (N = 21) patients. Mean cross-sectional area of PAD myofibers was reduced 29.3% compared to controls (p < 0.0003). Both forms of oxidative damage increased with clinical stage of disease, blood flow limitation in the ischemic leg, and reduced myofiber cross-sectional area. The data establish oxidative damage to myofibers as a possible cause of PAD myopathy.

Direct and indirect measurement of patient radiation exposure during endovascular aortic aneurysm repair.

With the increasing complexity of endovascular procedures, concern has grown regarding patient radiation exposure. Abdominal aortic aneurysm (AAA) repair represents the most common complex endovascular procedure currently performed by vascular specialists. Our study evaluates the patient radiation dose received during endovascular AAA repair. Over a 3-month period we prospectively monitored the radiation dose in a series of consecutive patients undergoing endovascular AAA repair. All patients underwent standard endovascular AAA repair with one of two commercially available grafts using the GE OEC 9800 unit. Direct measurement of maximum radiation dose at skin level (peak skin dose, PSD) was recorded using GAFCHROMIC radiographic dosimetry film. Indirect measurements of radiation dose (fluoroscopy time and dose-area-product [DAP]) were recorded with the C-arm dosimeter. A total of 12 consecutive patients undergoing standard endovascular AAA repair were evaluated. Mean PSD was 0.75 Gy (range 0.27-1.25). Mean total fluoroscopy time was 20.6 min (range 12.6-34.2) with an average of 92% spent in standard fluoroscopy and 8% spent in cinefluoroscopy. Regarding total fluoroscopy time, 49% was spent in normal field of view and 51% in magnified view. Mean DAP was 15,166 cGy x cm(2) (range 5,207-24,536). PSD correlated with DAP (r = 0.9, p < 0.05) but not total fluoroscopy time (r = 0.18, p > 0.05). PSD also correlated with body mass index (BMI; r = 0.82, p < 0.05). Obese patients had a mean PSD of 1.1 Gy compared to 0.5 Gy in nonobese patients. PSD of all patients was well below the accepted 2.0 Gy threshold for skin injury. PSD correlated with DAP but not total fluoroscopy time. PSD also correlated with BMI, and the mean PSD was significantly increased in obese compared to nonobese patients. Despite the complexity and duration of endovascular AAA repair, the procedure can be performed safely without excessive radiation exposure.

Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective.

BACKGROUND: Peripheral artery disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in the arteries supplying the legs, affects approximately 5% of Americans. We have previously, demonstrated that a myopathy characterized by myofiber oxidative damage and degeneration is central to PAD pathophysiology. OBJECTIVES: In this study, we hypothesized that increased oxidative damage in the myofibers of the gastrocnemius of PAD patients is myofiber-type selective and correlates with reduced myofiber size. METHODS: Needle biopsies were taken from the gastrocnemius of 53 PAD patients (28 with early PAD and 25 with advanced PAD) and 25 controls. Carbonyl groups (marker of oxidative damage), were quantified in myofibers of slide-mounted tissue, by quantitative fluorescence microscopy. Myofiber cross-sectional area was determined from sarcolemma labeled with wheat germ agglutinin. The tissues were also labeled for myosin I and II, permitting quantification of oxidative damage to and relative frequency of the different myofiber Types (Type I, Type II and mixed Type I/II myofibers). We compared PAD patients in early (N=28) vs. advanced (N=25) disease stage for selective, myofiber oxidative damage and altered morphometrics. RESULTS: The carbonyl content of gastrocnemius myofibers was higher in PAD patients compared to control subjects, for all three myofiber types (p<0.05). In PAD patients carbonyl content was higher (p<0.05) in Type II and I/II fibers compared to Type I fibers. Furthermore, the relative frequency and cross-sectional area of Type II fibers were lower, while the relative frequencies and cross-sectional area of Type I and Type I/II fibers were higher, in PAD compared to control gastrocnemius (p<0.05). Lastly, the type II-selective oxidative damage increased and myofiber size decreased as the disease progressed from the early to advanced stage. CONCLUSIONS: Our data confirm increased myofiber oxidative damage and reduced myofiber size in PAD gastrocnemius and demonstrate that the damage is selective for type II myofibers and is worse in the most advanced stage of PAD.

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage.

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.